Project Summary In this Project, we will investigate the dynamics of the host innate immune response induced by influenza A virus (IAV) in the main target cells of IAV replication in vivo: the human respiratory epithelial cells. For this purpose, we will compare induction and regulation of host responses upon infection of differentiated human tracheobronchial epithelial cells (HTBEs) with genotypically and phenotypically distinct IAV strains of human clinical relevance. Our project builds upon the previous characterization of these strains in monocyte-derived dendritic cells during our past NIAID contract-funded program for modeling immunity for biodefense (MIB) (PRiME). We now propose to elucidate the mechanisms underlying the immunological responses to IAV of the HTBEs. In Aim 1, the dynamics and special-temporal regulation of the host response induced by seasonal and pandemic H1N1 influenza virus strains will be studied using single cell technologies, such as CyTOF and RNA FISH assays. In concert with mathematical modeling in Project III, we will test the hypothesis that the innate immune responses and the transcriptional re-programming of HTBEs during IAV infection emerges from the interplay of several factors including differences among single cells, differences among IAV strains, and the autocrine and paracrine responses of the cells in response to the secreted cytokines. In Aim 2, we will determine how the host response varies according to the strength of IAV sensing, and to the ability of IAV to block host responses and to evade antiviral effectors of these responses. This will be done by probing HTBEs with mutant and reassortant IAV specifically differing in one of these parameters. This will also allow the validation (or further refinement) of the predictions of the model generated by Project III. In Aim 3, we will study the impact on HTBE innate immune responses of clinically relevant inhibitors of influenza virus replication working at different levels during the virus cycle. Responses in HTBEs will be compared with those in primary dendritic cells (studied in Project II), and validated in a human lung explants model (also studied in Project II). As the innate immune response in HTBEs is one of the first barriers against IAV infection, a better understanding of the regulation of the dynamics and magnitude of these responses and their impact in virus replication will help to better define what determines the outcome (virus inhibition versus unimpeded virus replication) of IAV infection.